Sequence comparison among short, homologous DNA molecules to reveal differences as slight as a single base pair substitution is an important step toward understanding the genetic basis for phenotypic variation and heritable disease.
When a DNA sequence from individual A differs from the DNA sequence of individual B by a single base pair substitution, one general approach to analyze such single base pair substitutions is to separate the strands from individuals A and B, mix and reanneal one strand from A and one strand B; because the sequences are identical except at the substituted site, complementary strands will reform the duplex DNA structure (since the strands are from two different individuals such a duplex is referred to herein as a heteroduplex). The heteroduplex will be a perfectly based paired DNA molecule except at the site of the base pair substitution. At this point a mismatch will occur. That is rather than the normal A-T, or G-C base pair, an A-C, A-G, T-C, T-G, A-A, G,G, T-T, C-C mispairing will be presented (see for example Page 11 infra). By detecting such an event one can better understand the nature of phenotypic variation and the heritable basis for certain diseases as well as providing a rational scheme for screening for such diseases.
Past efforts to detect mispaired bases include: (1) S.sub.1 and mung bean nuclease cleavage at the mismatched bases in heteroduplex DNA molecules (Shenk, T. E., et al, Proc. Nat'l. Acad. Sci. USA 72:989-993 (1975) and Kowalski, D., et al, Biochem 15:4457 (1976)) (these enzymes, however, cleave single base mismatches with relatively poor efficiency); (2) Changes in electrophoretic mobility in denaturing gradient polyacrylamide gels (Fisher, S. G. and Lerman, L. S., Proc. Nat'l. Acad. Sci. USA 80:1579-83 (1983)) (unless modified, this technique is able to detect only a fraction of differences in DNA base sequence); (3) Differential hybridization with oligonucleotide probes that differ by a single base (Wallace, R. B. et al Nucl. Acid Res 9:3647-56 (1981)) (this procedure is useful only if prior sequence information is available).
This invention overcomes the deficiencies of the prior art attempts to resolve single base pair mismatches by specifically "tagging" mismatched regions and detecting same electrophoretically. The invention also provides a method for purifying specific gene segments.